Resolution of neuropeptides by HPLC was studied as a function of mobile phase composition, flow rate, gradient rate, and the size and composition of the peptides themselves. Column efficiency in isocratic separations improved linearly with decreasing flow rate, and decreasing log-molecular weight of peptide. Resolution in gradient spearations was primarily a function of gradient rate; flow rate had little effect on resolution, but did affect peak height. The hypothesis that retention of peptides on HPLC could be predicted on the basis of amino acid consumption was tested using data from 100 peptides. The high correlation (0.98) indicates that conformation and sequence have minor effects on retention in comparison to the importance of composition.